DNA聚合酶适配子6-10提高定量PCR的特异性

Aptamer 6-10 of DNA polymerase can increase the sensitivity of quantitative PCR

目的利用DNA聚合酶适配子6-10进行热启动定量PCR,提高定量PCR的检测灵敏度。方法适配子6-10组、抗体对照组和非热启动对照组分别进行定量PCR,制作标准曲线,以复相关系数(r2)大于0.99为线性范围,比较其线性范围下限。结果非热启动定量PCR对人死亡受体5(death receptor 5,DR5)的检测线性范围下限为105拷贝/μl,而适配子6-10(200nmol/L)与抗DNA聚合酶抗体方法对DR5的检测线性范围下限为103拷贝/μl。熔解曲线分析表明,在扩增高浓度靶分子(105拷贝/μl)时,各种方法非特异扩增均不明显,没有形成非特异峰;在扩增低浓度靶分子(103拷贝/μl)时,非热启动对照组的非特异扩增明显,产生明显的非特异峰,而DNA聚合酶适配子6-10可以明显消除非特异扩增形成的非特异峰。结论DNA聚合酶适配子6-10可以提高定量PCR的检测灵敏度。

Objective To increase the sensitivity of quantitative PCR using DNA polymerase in combination with aptamer 6-10. Methods The quantitative PCR was performed using DNA polymerase mixed with aptamer 6-10 or specific antibody. The low detection limit of each modified method was compared with that of the conventional method. Results The quantitative PCR using aptamer 6-10 （200 nmol/L） or specific antibody increased the low detection limit of human death receptor 5 （DRS） plasmid from 105 copies/μl to 103 copies/μl. Melting curves showed that each method had minimal nonspecific amplification when the concentration of DR5 plasmid was 105 copies/μl; however, when the concentration of DR5 plasmid was 103 copies/μl, the conventional method had nonspecific amplification, whereas the method using aptamer 6-10 had minimal nonspecific amplification. Conclusions Aptamer 6-10 of DNA pelymerase can increase the sensitivity of quantitative PCR.